Hydroxytyrosol of olive oil: a promising bioactive compound to prevent hyperphosphorylation of tau protein in Alzheimer’sdisease
2024 – 2026 |Airalzh-Grants-for-Young-Researchers 2024
PI NICO Michela Guglielmotto
PI partner Gabriella Testa, Department of Clinical and Biological Sciences, Turin – Italy
High levels of hyperphosphorylated tau protein are linked with its accumulation in the form of neurofibrillary tangles (NFT) in Alzheimer’s disease (AD) pathogenesis. Growing evidence recognizes the very early onset of tau pathology and its key role in mediating amyloid-b (Ab) toxicity. Activation of tau degradation systems seems to be a very promising strategy for removing pathological tau during AD progression. Due to its ability to enter the brain tissue and high bioavailability along with its lack of toxicity, hydroxytyrosol (HXT), the most powerful polyphenol of extra virgin olive oil (EVOO), is considered a potential nutraceutical for AD prevention and treatment. Despite the recognized neuroprotective action of HXT against Aβ-induced toxicity, no studies about its effect in counteracting tau pathology are available. We demonstrated, in vitro, that HXT is a potent activator of the deacetylase SIRT-1, which plays a crucial role in regulating neuron survival because of its several beneficial effects in the brain, and that SIRT-1 activation promotes tau degradation through the Ubiquitin-Proteasome System (UPS).
However, to date, the effect of HXT on tau degradation via UPS activation remains unknown. The aim of this Project is to evaluate the beneficial effect of dietary supplementation of HXT on proteasomal degradation of tau protein in mice that express human tau (Mapt tm1(EGFP)KltTg(MAPT) 8cPdav/J; 005491 Jackson Laboratory). To generate an AD-like model, hTau mice were subjected to an intracerebroventricular (ICV) injection of okadaic acid (OKA), a useful Alzheimerogenic chemical that selectively inhibits the ability of serine/threonine phosphatase 2A (PP2A) to dephosphorylate tau resulting in tau hyperphosphorylation. Our goal will be to demonstrate that HXT-rich diet can prevent tau hyperphosphorylation induced by ICV injection of OKA, through SIRT-1-mediated proteasome activation. The protein levels of the pathological forms of tau (p-tau epitopes at residues Thr231, Ser202/Thr205 and Thr212/Ser214, and oligomeric tau), total tau, and acetylated and ubiquitinated tau, as well as SIRT-1 and proteasomal chymotrypsin-like activity, will be measured in the brain of mice fed with HXT and then treated with OKA and compared with mice treated only with OKA. Mouse cognitive behaviors will be also explored.HXT’s ability to counteract tau pathology could open new avenues in a hitherto unexplored field: dietary HXT supplementation could represent a winning strategy to avoid NFT formation, preventing the development of AD.
Neuroprotective role of the GHRH analog MR-409 in experimental models of Alzheimer’s disease
2023 – 2025 | BENEFICENTIA Stiftung Found
PI NICO Elena Tamagno
PI partner Riccarda Granata – Division of Endocrinology, Diabetology and Metabolism, University of Turin
Department of Medical Sciences, Turin – Italy
AD is a neurodegenerative disorder and the most common cause of dementia in elderly individuals, characterized by neuronal loss in the cerebral cortex, hippocampus, and amygdala. Clinical symptoms of AD include progressive memory decline, impaired executive function and difficulties executing routine daily activities. Mouse models of AD indicate that the level of neurogenesis is dramatically reduced in the early stage of the disease; proliferation and survival of newborn cells are significantly diminished during its progression. Thus, promoting neurogenesis has become a new therapeutic target for AD. Therapies to stimulate endogenous neurogenesis mainly include environmental stimulation, physical activity, neurotrophic factors, and drugs that may contribute to promoting regeneration and recovery processes. The main pathological features of AD are amyloid beta (Aβ) plaques and neurofibrillary tangles (NFTs), as well as neuroinflammation and oxidative stress in the brain. There is currently no effective treatment for AD, although there are available therapies that just improve the symptoms.The hypothalamic neurohormone growth hormone-releasing hormone (GHRH), in addition to promoting the release of pituitary growth hormone (GH), exerts many peripheral functions, such as the stimulation of cell survival and proliferation. We and others have demonstrated that both GHRH and its agonistic analogs, like MR-409, exert antinflammatory, cardioprotective and neuroprotective effects. Furthermore, our group has recently shown that MR-409 ameliorates the disease features of spinal muscular atrophy (SMA) mice, by improving muscular trophism, promoting the maturation of neuromuscular junctions and inhibiting neuroinflammation. Based on the foregoing, it is conceivable that GHRH may act as a positive regulator of neurogenesis and display neuroprotective effects. The role of full length GHRH(1-44)NH2 (from now on called GHRH) has been initially studied in vitro on survival, proliferation and differentiation of adult rat hippocampal neural stem cells (NSCs), which were found to express GHRH receptor (GHRH-R). Our preliminary results suggest that GHRH promotes neurogenesis and protects against Aβ-induced cell death. Specifically: 1) GHRH increased cell viability, promoted cell proliferation, and counteracted apoptosis of NSCs. These effects were observed in cells cultured under either growth factor deprivation or exposure to Aβ peptide (Aβ)1-42, a crucial driver of AD; 2) GHRH increased the differentiation of NSCs into neurons and astrocytes, as indicated by increased mRNA levels of the neuronal marker Tuj1 (neuron-specific class III beta-tubulin) and the astrocyte marker GFAP (glial fibrillary acidic protein). Evidence from the literature also indicate that MR-409 has remarkable neuroprotective effects through enhancing endogenous neurogenesis in cerebral ischemic mice. Furthermore, our group has previously demonstrated the proliferative and protective effects of growth hormone secretagogues, ghrelin and hexarelin, on adult rat hippocampal progenitors, while MR-409-induced protection against motor neuron cell death and neuroinflammation has been recently demonstrated in SMA mice. Based on the foregoing, the main aims of this project are: i) to determine whether GHRH promotes neurogenesis in vitro; ii) to assess the in vitro cytoprotective effect of GHRH against Aβ-induced toxicity, and iii) to explore the ability of the GHRH analog, MR-409, to improve the behavioral impairment and pathological features of AD in 5XFAD transgenic mice.
Study of early plasma markers of Alzheimer’sdisease: role of miRNA218
2024-2026 | CRT Foundation
PI Michela Guglielmotto
Alzheimer’s disease (AD) is the most common form of dementia and mainly afflicts the female population. The protective role of estrogens in preventing neurodegenerative diseases and neuroinflammation is well known, but the mechanisms by which they act remain unclear. Recently in our laboratories, using an animal model of disease, we demonstrated that there is a correlation between 17 ß-Estradiol (17 ß-E2) levels and miRNA-21 expression (1). The data were then clinically confirmed by analyzing plasma samples from both male and female, over 65 years of age, AD patients with mild cognitive impairment (MCI) and the control population. Our research project aims to identify a novel early plasma biomarker that will enable the patient and family to adhere to pharmacological and non pharmacological treatments aimed at fostering better quality of life and leading to reduced disability. In addition, using a novel mouse model developed in our laboratories: 5xTg- AD/hTauTg, we will investigate therapeutic approaches aimed at reducing the pathogenic effects of miRNA218 and controlling its estrogen-dependent modulation.
Extracellular vesicles derived from preconditioned Mesenchymal stem cell: a new challenge for AD therapy?
2023-2026 PRIN Ministry of University and Research
PI Elena Tamagno
Alzheimer’s disease (AD), the most common form of age-related dementia, is characterized by a progressive degeneration of the central nervous system (CNS) that leads to a gradualdecline of cognitive functions and memory loss. Recently, preclinical, epidemiological, and genetic studies have demonstrated in neurodegenerative diseases, including AD, an earlier involvement of immune system. Since there is still no cure for AD, these studies motivated the design of innovative therapeutic strategies aimingatslowing down degenerative processes by targeting not only neuronal but also glial cells, both microglia and astrocytes, in virtue of their main recognized role in orchestrating neuroinflammatory process. Mesenchymal stem cells (MSCs) are adultmultipotent stem cellsthat over the last decades have been demonstrated to convey improvement in various models of neurodegenerative pathologies, thanks to their paracrine ability that is largely dependent on the secretion of extracellular vesicles (EVs). The therapeutic potential of MSC-EVs, either as immuno modulators or as neuroprotective entities, has been recently put on focus also in the AD field. Moreover, the evidence that the intrinsec immunoregulatory abilities of MSCs is strongly influenced and strengthened by the environment, has led the scientists to design and optimize culture conditions (preconditioning) in order to enhance the anti-inflammatory properties of these cells and of their derived EVs. Thus, EVs produced by preconditioned human MSCs represent a promising therapeutic tool to limit or hamper neurodegeneration and inflammation in AD animal models. However, the neuroprotective and immunomodulatory mechanism striggered by preconditioned MSC-EVs are stillunclear and rather speculative. The present proposal aims to characterize the protective action of preconditioned MSC-EVs relatively to amyloid precursor protein (APP) processing and amyloid β (Aβ) formation, microtubulo associated protein tau (MAPT) phosphorylation and aggregation, inflammation and activation of glialcells, the crucial hallmarks of AD.
Selection and characterization of a new transgenic mouse model for the study of Alzheimer’s disease: 5xTg-AD/hTauTg
2023-2025 | Ricerca locale UNITO
PI Michela Guglielmotto
Co-PI Elena Tamagno
AlzheimeR’s disease (AD) is the most common form of senile degenerative dementia, causing an inexorable decline in cognitive and executive functions affecting memory, learning, and language.
Dementia is not a specific disease, but rather a general term that describes a wide range of symptoms associated with decline in memory or other intellectual abilities severe enough to
reduce an individual’s ability to perform daily activities independently. With the sporadic etiology of the disease predominating over the familial form, it is increasingly difficult to understand the underlying triggering event.
To date, research also focuses on lifestyle habits and those conditions that may be a risk factor that can over time terminate the disease or the possibility of using serum therapies that, however, neither prevent neuronal damage nor cure it. Infact, Alzheimer’s disease begins its asymptomatic course numerous years before cognitive deficits occur. The histological markers of the disease are senile plaques and neurofibrillary tangles consisting of amyloid protein and hyperphosphorylated tau protein, respectively. However, extensive studies focusing solely on the neurotoxicity of Aβ or tau have not shown significant efficacy in the treatment of the disease.
Therefore, focusing exclusively on the role of Aβ or only on the pathological conformational changes of tau while ignoring the interaction between Aβ and tau may not be entirely correct. In fact, Aβ and tau may interact via biologically active intermediate molecules, such as kinases like GSK-3β, CDK-5, and ERK. The pathological process likely involves an interaction between Aβ and tau in which these proteins amplify toxic effects, rather than a strictly hierarchical mode of interaction. Therefore, removal of Aβ or tau alone does not completely disrupt the interaction process, which continues to play a role in accelerating the pathological process (2,4). Therefore, suppression of the interaction may have more practical significance than simply focusing on the neurotoxicity of Aβ or tau alone. The possibility to study these mechanisms in vivo is offered by this new double transgenic that we want to breed and study. In this regard, there is no company or breeder that provides the model of the double transgenic that not only produces the Aβ protein endogenously but also has the human phenotype of unmutated tau (wild type).
The role of glymphatic system in sleep-dependent memory consolidation circuits
2023-2025 | Ricerca locale UNITO
PI Michela Guglielmotto
Recently, many studies correlate Alzheimer’s disease (AD) to sleep disorders. The effects of sleep disorders result in protein accumulation, due to the failure of the glymphatic system-dependent clearance. While several studies propose sleep deprivation as model of sleep disorders in mice, we recently validated on AD mouse model a protocol of sleep fragmentation (SF) which mimics more correctly a real condition of intermittent awakening commonly described in several sleep, psychiatric, and neurological disorders. SF alters NREM sleep and this is one of the hallmark signs of age-related changes in sleep5. In our experimental set up, we analyzed the effect of SF in 2-month-old 5xFAD and wild type (wt) mice. The animals were positioned for one month, 24 hours per day on a time-controlled tilting platform with an activation pattern of 3 minutes OFF/10 seconds ON. We observed a significant pathology acceleration in 5xFAD mice and an interesting aquaporin-4 (AQP4) modulation in both 5xFAD and wt mice.
In the light of these results, we believe that AQP4 modulation could be considered a risk factor for the disease development. As a consequence, we want to deepen our study focusing on a wt mouse model, in particular on the mechanisms responsible for the disruption of glymphatic system and the possible malfunction of hippocampal network. We have already observed (not published yet) a significant decrease in neuronal activation in the hippocampus of fragmented wt mice, in both young (2-month-old) and older group (6-month-old). Since the hippocampus is important for memory consolidation during NREM sleep6, we want to investigate which neuronal population is interested and possible neuronal death. To do this, we will immunostaining brain slices with the markers for two neuronal populations (v-Glut1, GABA or v-GAT), we will dedicate both control and fragmented mice of both ages for long-term potentiation studies, and we will correlate them with the results of behavioral tests for exploring memory capabilities.
As for the glymphatic system and its alterations, we will study through MRI the possible impairment in the glymphatic flux by using a paramagnetic contrast agent, such as Gd-DTPA, as MRI tracer. To have a better insight, the brain slices will be treated to be analyzed with electron microscopy (TEM) too. In this way, we will be able to observe the position in membrane of AQP4, which physiologically locates on the end-feet of astrocytes surrounding vessels to allow cerebrospinal fluid (CSF) and brain parenchyma exchanges (7,8), and to see whether astrocyte end-feet become hypertrophic, thus blocking the passage of CSF (3,7,9,10). A further step that we want to examine is to verify the possible rescue capability in young and older wt animals: the mice will be sacrificed one month after the end of the SF protocol.
In this way, we can observe whether young versus older animals are more capable of returning to their physiological condition. Thanks to this project, we back to the bench to
correlate the deficits in the hippocampal network, that lead to memory impairment after SF, to the possible involvement of glymphatic system. Since sleep disorders associated to an intermittent awakening are considered risk factors for AD (11), we can think of possible prevention or rescue strategies through the reactivation of the glymphatic system with AQP4 modulators or behavioral training that improve sleep quality and stimulates memory.
